UNIVERSITI PUTRA MALAYSIA LIPID PRODUCTION IN MARINE MICROALGAE UNDER DIFFERENT SALINITY, TEMPERATURE AND NUTRIENT LEVELS NURUL SALMA BINTI ADENAN IB 2012 16
LIPID PRODUCTION IN MARINE MICROALGAE UNDER DIFFERENT SALINITY, TEMPERATURE AND NUTRIENT LEVELS By NURUL SALMA BINTI ADENAN Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfillment of the Requirements for the Degree of Master of Science JULY 2012
Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment of the requirement for the degree of Master of Science LIPID PRODUCTION IN MARINE MICROALGAE UNDER DIFFERENT SALINITY, TEMPERATURE AND NUTRIENT LEVELS By NURUL SALMA BINTI ADENAN July 2012 Chairman: Professor Fatimah Md. Yusoff, PhD Institute: Bioscience The marine microalgae Chlorella sp. (UPMC-A0013) and Chaetoceros calcitrans (UPMC-A0010) contain relatively high lipid levels (15.0 to 20.0% of dry weight) that can contribute as important components for the formulation of feed in aquaculture industry. However, lipid levels in microalgae vary according to the culture conditions. This study was carried out to determine the various environmental factors that control the growth and lipid contents of marine Chlorella sp. and C. calcitrans by subjecting the algae to different levels of stress in terms of salinity and temperature changes and nutrient depletion in their early stationary phase. The effects of salinity stress on lipid production of Chlorella sp. and C. calcitrans were performed by culturing both microalgae species in Conway medium of various salinity levels (15, 20, 25, 30, 35 and 40 ). The cultures were centrifuged and resuspended into culture medium of lower salinity levels of -5 (S1 i ) and -10 ii
(S2 i ) from the initial salinity levels in their early stationary phase. The highest growth rate (P<0.05) of Chlorella sp. and C. calcitrans were observed at 25 (μ=0.37 day -1 ) and 30 (μ=0.28 day -1 ), respectively. Salinity changes from S 30 to S1 30 and S2 30 increased P<0.05) the production of total lipid in Chlorella sp. to 10.0% and 19.0% of d.w., respectively. Total lipid of C. calcitrans increased significantly (P<0.05) to 13.2% of d.w. when stressed to S2 30. Prior to the temperature stress, marine Chlorella sp. and C. calcitrans were cultivated at 20, 25, 30 and 35 o C (T i ). Then, the microalgae were shifted to higher temperature of +5 o C (TS i ) from the initial temperature levels in their early stationary phase. Before stress, Chlorella sp. showed the highest growth rate (µ= 0.35 day -1, P<0.05) at 25 o C (T 25 ). Total lipid of Chlorella sp. significantly increase (P<0.05) from 31.0% of d.w. (T 25 ) to 41.0% of d.w (TS 25 ). Diatom, C. calcitrans showed the highest growth rate at T 30 (µ= 0.26 day -1 ) and lipid content increased significantly (P<0.05) from 31.0% to 39.0% after stress (TS 30 ). The effects of nutrient stress on lipid production was determined by culturing the algae into Nitrogen and Phosphorus deprived Conway medium (25.0% reduction = N 25 and P 25 ; 50.0% reduction = N 50 and P 50 ; 75.0% reduction = N 75 and P 75 ) in their early stationary phase. Lipid concentrations significantly increased (P<0.05) by 9.0% in both cultures subjected to 75% nitrogen and phosphorus reduction (N 75 and P 75 ). This study illustrated that various growth conditions such as salinity and temperature changes, and reduction in nutrient concentration enhanced lipid content of Chlorella sp. and C. calcitrans. iii
Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Sarjana Sains PENGHASILAN LIPID OLEH MIKROALGA MARIN DI BAWAH TAHAP KEMASINAN, SUHU DAN NUTRIEN BERBEZA Oleh NURUL SALMA BINTI ADENAN Julai 2012 Pengerusi : Professor Fatimah Md. Yusoff, PhD Institut : Biosains marin mikroalga, Chlorella sp. (UPMC-AA0013) dan C. calcitrans (UPMC- AA0010) mengandungi kadar lemak yang tinggi (15-20% berat kering) yang boleh diaplikasikan sebagai komponen penting dalam pembuatan dan penghasilan makanan bagi industri akuakultur. Namun begitu, kadar lipid yang dihasilkan bergantung kepada kaedah pengkulturan. Dengan itu, penentuan pelbagai faktor pengkulturan yang boleh mengawal kadar pertumbuhan dan penghasilan lipid adalah penting. Kadar pertumbuhan dan penghasilan lipid oleh Chlorella sp. dan C. calcitrans dilakukan dengan mengaplikasikan beberapa tahap tekanan pada pembolehubah pertumbuhan, yang merangkumi perubahan tahap kemasinan dan suhu, serta pengurangan nutrisi di awal fasa pertumbuhan mendatar. iv
Kajian kesan perubahan tahap kemasinan terhadap pengeluaran lipid bagi Chlorella sp. dan C. calcitrans dengan mengkultur kedua-dua mikroalga di dalam media Conway yang mengandungi tahap kemasinan asal (S i ) yang berbeza iaitu 15, 20, 25, 30, 35 dan 40. Kultur diemparkan dan dilarutkan ke dalam media yang mempunyai tahap kemasinan yang rendah, -5 (S1 i ) dan -10 (S2 i ) dari kepekatan asal. Chlorella sp. dan C. calcitrans menunjukkan kadar pertumbuhan yang tinggi pada 25 (μ = 0.37 hari -1 ) dan 30 (μ=0.28 hari -1 ). Chlorella sp. menunjukkan peningkatan pengeluaran lipid kepada 10.0% and 19.0% berat kering setelah berlaku perubahan tahap kemasinan dari S 30 to S1 30 dan S2 30. Jumlah pengeluaran lipid bagi C. calcitrans meningkat kepada 13.2% berat kering setelah tahap kemasinan diubah ke S2 30. Chlorella sp. dan C. calcitrans dikultur pada suhu 20, 25, 30 and 35 o C. Kemudian, peningkatan suhu sebanyak +5 o C (TS i ) dari suhu asal (T i ) dilakukan pada peringkat awal fasa pertumbuhan mendatar. Kadar pertumbuhan Chlorella sp. adalah µ= 0.35 day -1 (P<0.05) pada suhu 25 o C sebelum suhu dinaikkan. Kadar lipid meningkat dari 31.0% berat kering pada TS 25 ke 41.0% berat kering selepas suhu dinaikkan ke 30 o C (TS 25 ). Chaetoceros calcitrans menunjukkan kadar pertumbuhan yang tinggi pada suhu T 30 (µ= 0.26 hari -1 ). Selepas suhu dinaikkan, kadar lipid meningkat dari 31.0% ke 39.0% berat kering pada TS 30. Dalam menentukan kesan pengurangan nutrisi terhadap kadar penghasilan lemak, Chlorella sp. marin dan C. calcitrans dikultur ke dalam media Conway dan dilarutkan ke dalam kultur media yang mengandungi beberapa siri kepekatan v
nitrogen dan fosforus yang dikurangkan (pengurangan 25.0% = N 25 dan P 25 ; pengurangan 50.0% = N 50 dan P 50 ; pengurangan 75.0% = N 75 dan P 75 ) pada awal fasa pertumbuhan mendatar. Peratusan lipid bagi kedua-dua spesis didapati meningkat sebanyak 9.0% berat kering apabila kepekatan nitrogen dan fosforus dikurangkan sehingga 75.0%. Kajian ini menunjukkan perubahan tahap kemasinan dan suhu serta pengurangan kadar nutrisi dapat meningkatkan penghasilan peratusan lipid yang tinggi dalam Chlorella sp. marin dan C. calcitrans. vi
ACKNOWLEDGEMENTS Assalamualaikum, First of all thank you Allah, for your love and guidance I have successfully completed my laboratory work and finally, this thesis. My sincere gratitude goes to my supervisors, Prof. Dr Fatimah Md. Yusoff and Prof. Dato Dr Mohamed Shariff Mohamed Din for your guidance and encouragement throughout the research. Special thanks also extended to all my colleagues for their assistance, encouragement and friendship, I love you guys. To lecturers, researchers and science officers who were involved directly or indirectly throughout research, thank you for you help and advice. Last but not least, I would like to acknowledge with thank and love to my family and husband for all their love, support and the tremendous efforts they have put to materialize this thesis. vii
I certify that an Examination Committee has met on 26 th July 2012 to conduct the final examination of Nurul Salma Binti Adenan on her Master in Science thesis entitled Lipid Production In Marine Microalgae Under Different Salinity, Temperature And Nutrient Levels in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980. The committee recommends that the student be awarded the Master of Science. Members of the Examination committee were as follows: Aziz Arshad, PhD Professor Faculty of Agriculture Universiti Putra Malaysia (Chairman) Mohamad Pauzi Zakaria, PhD Professor Faculty of Environmental Science Universiti Putra Malaysia (Internal examiner) Che Roos Saad, PhD Associate Professor Faculty Agriculture Universiti Putra Malaysia (Internal examiner) Wan Maznah Wan Omar, PhD Associate Professor School of Biological Sciences Universiti Sains Malaysia Malaysia (External examiner) SEOW HENG FONG Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date: viii
This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfillment of the requirement for the degree of Master of Science. The members of the Supervisory Committee were as follows: Fatimah binti Md. Yusoff, PhD Professor Institute of Bioscience Universiti Putra Malaysia (Chairman) Mohamed Shariff bin Mohamed Din, PhD Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Member) BUJANG BIN KIM HUAT, PhD Professor and Dean School of Graduate Studies Universiti Putra Malaysia Date: ix
DECLARATION I declare that the thesis is my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously, and is not concurrently, submitted for any other degree at Universiti Putra Malaysia or at any other institution. NURUL SALMA BINTI ADENAN Date: 26 July 2012 x
TABLE OF CONTENTS ABSTRACT ii ABSTRAK iv ACKNOWLEDGEMENTS vii APPROVAL DECLARATION TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS CHAPTER 1 INTRODUCTION 1.1 Background of study 1 1.2 Problem statement 3 1.3 Objectives 4 2 LITERATURE REVIEW 2.1 Biology and distributions of microalgae 5 2.2 Application of microalgae 7 2.3 History of microalgae in aquaculture industry 8 2.4 Bioactive compounds of microalgae 10 2.5 Fatty acid proportions in microalgae 11 2.6 Microalgae as a source of beneficial nutrients for aquaculture 2.7 Lipid biosynthesis in microalgae 14 2.8 Factors influencing the increment of microalgae lipid viii production 2.8.1 Salinity 16 2.8.2 Temperature 17 2.8.3 Nitrogen deprivation 18 2.8.4 Phosphorus deprivation 19 x xi xiv xvi xix 13 15 xi
3 GENERAL METHODOLOGY 3.1 Algal cultures 21 3.1.1 Cultural condition of the starter culture 23 3.2 Microalgae identification 23 3.2.1 Light microscope observation 23 3.2.2 Molecular DNA identification equal 24 3.2.2.1 Polymerase chain reaction and sequencing 25 3.3 Experimental Design 26 3.4 Statistical Analyses 27 3.5 Determination of cell concentration 29 3.5.1 Optical density 29 3.5.2 Cell counts 29 3.5.3 Biomass estimation 30 3.5.4 Estimation of growth rate 30 3.6 Biochemical analysis 31 3.6.1 Total Lipid Determination 31 3.6.2 Determination of fatty acids 32 3.6.3 Proximate analysis 33 3.6.3.1 Determination of protein 34 3.6.3.2 Determination of carbohydrate 35 4 EFFECTS OF DIFFERENT SALINITY LEVELS ON LIPID PRODUCTION OF MARINE MICROALGAE 4.1 Introduction 36 4.2 Materials and methods 37 4.2.1 Experimental design 37 4.2.2 Algal production 39 4.3 Results 39 4.4 Discussion 52 xii
5 EFFECTS OF DIFFERENT TEMPERATURE LEVELS ON LIPID PRODUCTION OF MARINE MICROALGAE 5.1 Introduction 57 5.2 Materials and methods 58 5.2.1 Experimental design 58 5.2.2 Algal production 59 5.3 Results 59 5.4 Discussion 71 6 EFFECTS OF NITROGEN REDUCTION ON LIPID PRODUCTION OF MARINE MICROALGAE 6.1 Introduction 76 6.2 Materials and methods 77 6.2.1 Experimental design 77 6.2.2 Algal production 79 6.3 Results 79 6.4 Discussion 87 7 EFFECTS OF PHOSPHORUS REDUCTION ON LIPID PRODUCTION OF MARINE MICROALGAE 7.1 Introduction 93 7.2 Materials and methods 94 7.2.1 Experimental design 94 7.2.2 Algal production 96 7.3 Results 96 7.4 Discussion 105 8 SUMMARY, GENERAL CONCLUSION & RECOMMENDATION REFERENCES 113 APPENDICES 134 BIODATA OF STUDENT 141 109 xiii